Impact of Schistosoma japonicum infection on collagen-induced arthritis in DBA/1 mice: a murine model of human rheumatoid arthritis

Background

The hygiene hypothesis suggests that helminth infections prevent a range of autoimmune diseases.

Methodology/Principal Findings

To investigate the effects of S. japonicum infection on collagen-induced arthritis (CIA), male DBA/1 mice were challenged with unisexual or bisexual S. japonicum cercariae two weeks prior to bovine type II collagen (CII) immunization or at the onset of CIA. S. japonicum infection prior to CII immunization significantly reduced the severity of CIA. ELISA (enzyme linked immunosorbent assay) showed that the levels of anti-CII IgG and IgG2a were reduced in prior schistosome-infected mice, while anti-CII IgG1 was elevated. Splenocyte proliferation against both polyclonal and antigen-specific stimuli was reduced by prior schistosome infection as measured by tritiated thymidine incorporation (³H-TdR). Cytokine profiles and CD4⁺ T cells subpopulation analysis by ELISA and flow cytometry (FCM) demonstrated that prior schistosome infection resulted in a significant down-regulation of pro-inflammatory cytokines (IFN-γ, TNF-α, IL-1β and IL-6) and Th1 cells, together with up-regulation of the anti-inflammatory cytokine IL-10 and Th2 cells. Interestingly, the expansion of Treg cells and the reduction of Th17 cells were only observed in bisexually infected mice. In addition, prior schistosome infection notably reduced the expression of pro-inflammatory cytokines and receptor activator of NF-κB ligand (RANKL) in the inflamed joint. However, the disease was exacerbated at one week after infection when established CIA mice were challenged with bisexual cercariae.

Conclusion/Significance

Our data provide direct evidence that the Th2 response evoked by prior S. japonicum infection can suppress the Th1 response and pro-inflammatory mediator and that bisexual infection with egg-laying up-regulates the Treg response and down-regulates the Th17 response, resulting in an amelioration of autoimmune arthritis. The beneficial effects might depend on the establishment of a Th2-dominant response rather than the presence of the eggs. Our results suggest that anti-inflammatory molecules from the parasite could treat autoimmune diseases.     

Gait analysis in a murine model of collagen-induced arthritis

Murine collagen-induced arthritis (CIA) has become a valuable animal model for elucidating pathogenic mechanisms and evaluating therapeutic effects for rheumatoid arthritis. Recent advances in digital imaging and computer technology have enabled gait analysis to develop into a powerful tool for objectively detecting functional deficits in human and animal models. The present study explored the use of non-invasive video-capture gait analysis in the evaluation of a murine CIA model. CIA was induced in 45 female DBA/1LacJ mice (8 to 10 weeks old) by immunization with lyophilized bovine articular type II collagen. Gait parameters were determined by ventral plane videography and were correlated to traditional arthritis clinical scores. Our results showed that increases in clinical scores that measure the severity of CIA corresponded to changes in multiple gait parameters that reflect both morphologic (increases in paw area) and functional (increase in stride frequency, decrease in stride length, hind-limb paw placement angle, as well as stride, stance, and braking times) deficits. Our work indicated that the non-invasive video-capture device may be used as a simple and objective data acquisition system for quantifying gait disturbances in CIA mice for the investigation of mechanisms and the evaluation of therapeutic agents.     

The inhibitory effects of a RANKL-binding peptide on articular and periarticular bone loss in a murine model of collagen-induced arthritis: a bone histomorphometric study

IntroductionWe designed OP3-4 (YCEIEFCYLIR), a cyclic peptide, to mimic the soluble osteoprotegerin (OPG), and was proven to bind to RANKL (receptor activator of NF-κB ligand), thereby inhibiting osteoclastogenesis. We recently found that another RANKL binding peptide, W9, could accelerate bone formation by affecting RANKL signaling in osteoblasts. We herein demonstrate the effects of OP3-4 on bone formation and bone loss in a murine model of rheumatoid arthritis.MethodsTwenty-four seven-week-old male DBA/1J mice were used to generate a murine model of collagen-induced arthritis (CIA). Then, vehicle or OP3-4 (9 mg/kg/day or 18 mg/kg/day) was subcutaneously infused using infusion pumps for three weeks beginning seven days after the second immunization. The arthritis score was assessed, and the mice were sacrificed on day 49. Thereafter, radiographic, histological and biochemical analyses were performed.ResultsThe OP3-4 treatment did not significantly inhibit the CIA-induced arthritis, but limited bone loss. Micro-CT images and quantitative measurements of the bone mineral density revealed that 18 mg/kg/day OP3-4 prevented the CIA-induced bone loss at both articular and periarticular sites of tibiae. As expected, OP3-4 significantly reduced the CIA-induced serum CTX levels, a marker of bone resorption. Interestingly, the bone histomorphometric analyses using undecalcified sections showed that OP3-4 prevented the CIA-induced reduction of bone formation-related parameters at the periarticular sites.ConclusionThe peptide that mimicked OPG prevented inflammatory bone loss by inhibiting bone resorption and stimulating bone formation. It could therefore be a useful template for the development of small molecule drugs for inflammatory bone loss.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0753-8) contains supplementary material, which is available to authorized users.     

Treatment with recombinant interferon-beta reduces inflammation and slows cartilage destruction in the collagen-induced arthritis model of rheumatoid arthritis

We investigated the therapeutic potential and mechanism of action of IFN-beta protein for the treatment of rheumatoid arthritis (RA). Collagen-induced arthritis was induced in DBA/1 mice. At the first clinical sign of disease, mice were given daily injections of recombinant mouse IFN-beta or saline for 7 days. Disease progression was monitored by visual clinical scoring and measurement of paw swelling. Inflammation and joint destruction were assessed histologically 8 days after the onset of arthritis. Proteoglycan depletion was determined by safranin O staining. Expression of cytokines, receptor activator of NF-kappaB ligand, and c-Fos was evaluated immunohistochemically. The IL-1-induced expression of IL-6, IL-8, and granulocyte/macrophage-colony-stimulating factor (GM-CSF) was studied by ELISA in supernatant of RA and osteoarthritis fibroblast-like synoviocytes incubated with IFN-beta. We also examined the effect of IFN-beta on NF-kappaB activity. IFN-beta, at 0.25 microg/injection and higher, significantly reduced disease severity in two experiments, each using 8-10 mice per treatment group. IFN-beta-treated animals displayed significantly less cartilage and bone destruction than controls, paralleled by a decreased number of positive cells of two gene products required for osteoclastogenesis, receptor activator of NF-kappaB ligand and c-Fos. Tumor necrosis factor alpha and IL-6 expression were significantly reduced, while IL-10 production was increased after IFN-beta treatment. IFN-beta reduced expression of IL-6, IL-8, and GM-CSF in RA and osteoarthritis fibroblast-like synoviocytes, correlating with reduced NF-kappaB activity. The data support the view that IFN-beta is a potential therapy for RA that might help to diminish both joint inflammation and destruction by cytokine modulation.     

Age-related changes in arthritis susceptibility and severity in a murine model of rheumatoid arthritis

Background

Rheumatoid arthritis (RA) most often begins in females in the fourth-fifth decade of their life, suggesting that the aging of the immune system (immunosenescence) has a major role in this disease. Therefore, in the present study, we sought to investigate the effect of age on arthritis susceptibility in BALB/c mice using the proteoglycan (PG)-induced arthritis (PGIA) model of RA.

Results

We have found that young, 1-month-old female BALB/c mice are resistant to the induction of PGIA, but with aging they become susceptible. PG-induced T cell responses decline with age, whereas there is a shift toward Th1 cytokines. An age-dependent decrease in T cell number is associated with an increased ratio of the memory phenotype, and lower CD28 expression. Antigen-presenting cells shifted from macrophages and myeloid dendritic cells in young mice toward B cells in older mice. The regulatory/activated T cell ratio decreases in older mice after PG injections indicating impaired regulation of the immune response.

Conclusion

We conclude that immunosenescence could alter arthritis susceptibility in a very complex manner including both adaptive and innate immunities, and it cannot be determined by a single trait. Cumulative alterations in immunoregulatory functions closely resemble human disease, which makes this systemic autoimmune arthritis model of RA even more valuable.     

A novel therapy of murine collagen-induced arthritis with soluble T1/ST2

Rheumatoid arthritis is characterized by chronic inflammatory infiltration of the synovium, leading to eventual cartilage and bone destruction. Previously, we have reported that soluble T1/ST2 (sST2), a member of the IL-1R gene family, inhibits LPS-induced macrophage proinflammatory cytokine production. In this study, we report the therapeutic effect of sST2-Fc in the murine model of collagen-induced arthritis. A short term administration of sST2-Fc fusion protein significantly attenuated disease severity compared with controls treated with normal IgG. Histological examination revealed that while control IgG-treated mice developed severe cellular infiltration in the joints, synovial hyperplasia, and joint erosion, this pathology was profoundly reduced in sST2-Fc-treated animals. Treatment of sST2-Fc also down-regulated serum levels of IL-6, IL-12, and TNF-alpha. Spleen cells from the sST2-Fc-treated mice produced significantly less IFN-gamma, TNF-alpha, IL-6, and IL-12 compared with cells from the control mice when cultured with collagen in vitro. Finally, pretreatment with ST2-Fc markedly inhibited the ability of human monocytic THP1 cells to release TNF-alpha when cocultured with peripheral blood T cells from rheumatoid patients. Together these results demonstrate that sST2-Fc may provide a novel approach in treating chronic autoimmune conditions by inhibiting the release of proinflammatory cytokines.     

Activation of formyl peptide receptor 1 elicits therapeutic effects against collagen-induced arthritis

Rheumatoid arthritis (RA) is an autoimmune disorder which shows production of autoantibodies, inflammation, bone erosion, swelling and pain in joints. In this study, we examined the effects of an immune-modulating peptide, WKYMVm, that is an agonist for formyl peptide receptors (FPRs). Administration of WKYMVm into collagen-induced arthritis (CIA) mice, an animal model for RA, attenuated paw thickness, clinical scores, production of type II collagen-specific antibodies and inflammatory cytokines. WKYMVm treatment also decreased the numbers of T_H1 and T_H17 cells in the spleens of CIA mice. WKYMVm attenuated T_H1 and T_H17 differentiation in a dendritic cell (DC)-dependent manner. WKYMVm-induced beneficial effects against CIA and WKYMVm-attenuated T_H1 and T_H17 differentiation were reversed by cyclosporin H but not by WRW4, indicating a crucial role of FPR1. We also found that WKYMVm augmented IL-10 production from lipopolysaccharide-stimulated DCs and WKYMVm failed to suppress T_H1 and T_H17 differentiation in the presence of anti-IL-10 antibody. The therapeutic administration of WKYMVm also elicited beneficial outcome against CIA. Collectively, we demonstrate that WKYMVm stimulation of FPR1 in DCs suppresses the generation of T_H1 and T_H17 cells via IL-10 production, providing novel insight into the function of FPR1 in regulating CIA pathogenesis.     

Effect of sclerostin-neutralising antibody on periarticular and systemic bone in a murine model of rheumatoid arthritis: a microCT study

Introduction

Patients with chronic inflammatory diseases have increased bone loss and bone fragility and are at increased risk of fracture. Although anti-resorptive drugs are effective in blocking inflammation-induced bone loss, they are less effective at rebuilding bone. We have previously shown that treatment with sclerostin antibody (Scl-AbI) builds bone and can prevent or restore bone loss in a murine model of inflammatory bowel disease. In this study, we tested the effect of Scl-AbI in a murine model of rheumatoid arthritis (the collagen-induced arthritis model, CIA). We hypothesised that sclerostin blockade can protect and restore bone both locally and systemically without affecting progression of inflammation.

Methods

CIA was induced in male DBA/1 mice, which were treated with either PBS or Scl-AbI (10 mg/kg, weekly) prophylactically for 55 days or therapeutically for 21 days (starting 14 days post onset of arthritis). Systemic inflammation was assessed by measuring the serum concentration of anti-CII IgG1, IgG2a and IgG2b by ELISA. Changes in bone mass and structure, either at sites remote from the joints or at periarticular sites, were measured using DEXA and microCT. Bone focal erosion was assessed in microCT scans of ankle and knee joints.

Results

Circulating anti-CII immunoglobulins were significantly elevated in mice with CIA and there were no significant differences in the levels of anti-CII immunoglobulins in mice treated with PBS or Scl-ABI. Prophylactic Scl-AbI treatment prevented the decrease in whole body bone mineral density (BMD) and in the bone volume fraction at axial (vertebral body) and appendicular (tibial proximal metaphysis trabecular and mid-diaphysis cortical bone) sites seen in PBS-treated CIA mice, but did not prevent the formation of focal bone erosions on the periarticular bone in the knee and ankle joints. In the therapeutic study, Scl-AbI restored BMD and bone volume fraction at all assessed sites but was unable to repair focal erosions.

Conclusions

Sclerostin blockade prevented or reversed the decrease in axial and appendicular bone mass in the murine model of rheumatoid arthritis, but did not affect systemic inflammation and was unable to prevent or repair local focal erosion.     

A role for the lymphotoxin/LIGHT axis in the pathogenesis of murine collagen-induced arthritis

A lymphotoxin-beta (LTbeta) receptor-Ig fusion protein (LTbetaR-Ig) was used to evaluate the importance of the lymphotoxin/LIGHT axis in the development and perpetuation of arthritis. Prophylactic treatment with the inhibitor protein LTbetaR-Ig blocked the induction of collagen-induced arthritis in mice and adjuvant arthritis in Lewis rats. Treatment of mice with established collagen-induced arthritis reduced the severity of arthritic symptoms and joint tissue damage. However, in a passive model of anti-collagen Ab-triggered arthritis, joint inflammation was not affected by LTbetaR-Ig treatment precluding LT/LIGHT involvement in the very terminal immune complex/complement/FcR-mediated effector phase. Collagen-II and Mycobacterium-specific T cell responses were not impaired, yet there was evidence that the overall response to the mycobacterium was blunted. Serum titers of anti-collagen-II Abs were reduced especially during the late phase of disease. Treatment with LTbetaR-Ig ablated follicular dendritic cell networks in the draining lymph nodes, suggesting that impaired class switching and affinity maturation may have led to a decreased level of pathological autoantibodies. These data are consistent with a model in which the LT/LIGHT axis controls microenvironments in the draining lymph nodes. These environments are critical in shaping the adjuvant-driven initiating events that impact the subsequent quality of the anti-collagen response in the later phases. Consequently, blockade of the LT/LIGHT axis may represent a novel approach to the treatment of autoimmune diseases such as rheumatoid arthritis that involve both T cell and Ab components.     

Discoidin domain receptor 1 mediates collagen-induced nitric oxide production in J774A.1 murine macrophages

Nitric oxide (NO) is an important regulator of immune responses. Effects of cytokines, such as tumor necrosis factor (TNF)-alpha or IFN-gamma, and bacterial products, such as lipopolysaccharide, on macrophage NO production have been well documented; however, the role of the extracellular matrix proteins, including collagen, in this process remains unclear. We previously reported that discoidin domain receptor 1 (DDR1), a nonintegrin collagen receptor, was expressed in human macrophages, and its activation facilitated their differentiation as well as cytokine/chemokine production. Here, we examined the role for DDR1 in collagen-induced NO production using the murine macrophage cell line J774 cells that endogenously express DDR1. Activation of J774 cells with collagen induced the expression of inducible NO synthase (iNOS) and NO production. Inhibition of DDR1, but not beta1-integrins, abolished collagen-induced iNOS and NO production. Activation of J774 cells with collagen-activated nuclear factor-kappaB, p38 mitogen-activated protein kinase (MAPK), and c-jun N-terminal kinase (JNK) and a pharmacological inhibitor of each signaling molecule significantly reduced collagen-induced NO production. Thus, we have demonstrated, for the first time, that the interaction of DDR1 with collagen induces iNOS expression and subsequent NO synthesis in J774 cells through activation of NF-kappaB, p38 MAPK, and JNK and suggest that intervention of DDR1 signaling in macrophages may be useful in controlling inflammatory diseases in which NO plays a critical role.     

The angiogenesis inhibitor protease-activated kringles 1-5 reduces the severity of murine collagen-induced arthritis

During rheumatoid arthritis there is enlargement and increased cellularity of the synovial lining of joints, before invasion by the synovium of the underlying cartilage and bone. This increased tissue mass requires a network of blood vessels to supply nutrients and oxygen. Disruption of synovial angiogenesis is thus a desirable aim of antiarthritic therapies. Protease-activated kringles 1-5 (K1-5) is an angiogenesis inhibitor related to angiostatin. In common with angiostatin, K1-5 contains the first four kringle domains of plasminogen, but also encompasses the kringle 5 domain, which confers enhanced antiangiogenic activity when compared with angiostatin. The purpose of the present study was to assess the effect on murine arthritis of K1-5. Arthritis was induced in DBA/1 mice by a single injection of bovine collagen. Treatment with K1-5 was commenced on the day of arthritis onset and continued for 10 days, until the end of the experiment. Daily intraperitoneal administration of K1-5 (2 mg/kg body weight) significantly reduced both paw swelling and clinical score (a composite index of the number of arthritic limbs and the severity of disease). The clinical efficacy of this treatment was reflected by a reduction in joint inflammation and destruction, as assessed histologically. These data suggest that antiangiogenic therapies, which block formation of new blood vessels and hence reduce synovial expansion, might be effective in treating rheumatoid arthritis.     

Adenoviral transfer of cyclin-dependent kinase inhibitor genes suppresses collagen-induced arthritis in mice

In rheumatoid synovial tissues, synovial fibroblasts are activated by proinflammatory cytokines and proliferate to develop hyperplastic pannus tissues, which irreversibly damage the affected joints. We recently reported that the cyclin-dependent kinase inhibitors p16(INK4a) and p21(Cip1) are not expressed in vivo in rheumatoid synovial fibroblasts, but are readily inducible in vitro. This observation was followed by the successful treatment of rat adjuvant arthritis by local p16(INK4a) gene transfer, showing that the inhibition of the cell cycle of the synovial cells ameliorates the arthritis. In this study, we show that another animal model of rheumatoid arthritis, murine collagen-induced arthritis, can be effectively treated by local gene transfer of p21(Cip1) as well as that of p16(INK4a). The anti-arthritic effects were observed even when the treatment was conducted after the arthritis had developed. Furthermore, the effects included suppression of the expression of proinflammatory cytokines such as IL-1ss, IL-6, and TNF-alpha. Our results demonstrate that the ectopic expression of cyclin-dependent kinase inhibitors not only prevents synovial overgrowth but also ameliorates the proinflammatory milieu in the affected joints. The induction of p21(Cip1) in rheumatoid synovial tissues by pharmacological agents may also be an effective strategy to treat rheumatoid arthritis.     

Electrotransfer of human IL-1Ra into skeletal muscles reduces the incidence of murine collagen-induced arthritis

BACKGROUND: It has previously been demonstrated that high levels of gene expression in skeletal muscles can be achieved after direct in vivo electrotransfer of naked plasmid DNA. The purpose of this study is to examine the potential of in vivo electroporation of plasmid DNA encoding human IL-1Ra for the prevention of murine collagen-induced arthritis (CIA). METHODS: DBA/1 mice were injected in gastrocnemius muscles with plasmid DNA followed by in vivo electroporation. To uncover the optimum conditions of gene transfer, various electric field strengths and different amounts of plasmid DNA were applied. Calf muscles around the injected areas were investigated with histological methods for damage to muscle tissue. The levels of human IL-1Ra expression in the injected area and also in the serum were determined with ELISA for human IL-1Ra. Based on these data, the effects of electrotransfer of plasmid DNA were tested using the murine CIA model. DBA/1 mice were immunized with bovine collagen type II at the base of the tail. On day 21, mice were given a booster injection with the same antigen. Mice were divided into two groups on day 26. One group of mice received plasmid containing the IL-1Ra cDNA sequence, while control mice were given plasmid lacking the IL-1Ra coding sequence. The incidence of arthritis was evaluated by macroscopic analysis, histological analysis, and the levels of inflammatory cytokines. RESULTS: IL-1Ra expression increased as a function of the electrical field strength and the amount of DNA. 200 V/cm (eight pulses; 20 ms per pulse; 1 Hz) and 15 microg of plasmid DNA per mouse were found to be optimum for gene transfer. After in vivo electroporation, gene expression in both muscle and serum increased gradually, reaching a peak value on day 10. Significant levels of human IL-1Ra expression were maintained for 20 days. Macroscopic analysis showed that the onset of CIA was significantly inhibited by direct electrotransfer of plasmid DNA encoding human IL-1Ra. Histological analysis of knee joints showed that the incidence of arthritis in knee joints was also prevented. The levels of mouse IL-1beta and IL-12 in paws were significantly lower in the group treated with IL-1Ra than those in the control group. CONCLUSIONS: These results demonstrate that direct electrotransfer of plasmid containing the human IL-1Ra cDNA sequence to skeletal muscle can reduce the incidence of CIA in mice.     

Vascular endothelial growth factor expression and regulation of murine collagen-induced arthritis

We have examined the expression and function of the angiogenic factor, vascular endothelial growth factor (VEGF) during the evolution of type II collagen-induced arthritis (CIA). Biologically active VEGF was expressed along a time course that paralleled the expression of two specific VEGF receptors, Flk-1 and Flt-1, and the progression of joint disease. Moreover, levels of VEGF expression correlated with the degree of neovascularization, as defined by vWF levels, and arthritis severity. Macrophage- and fibroblast-like cells, which infiltrated inflamed sites and were then activated by other inflammatory mediators, are probably important sources of VEGF and may thus regulate angiogenesis during the development of CIA. Administration of anti-VEGF antiserum to CIA mice before the onset of arthritis delayed the onset, reduced the severity, and diminished the vWF content of arthritic joints. By contrast, administration of anti-VEGF antiserum after the onset of the disease had no effect on the progression or ultimate severity of the arthritis. These data suggest that VEGF plays a crucial role during an early stage of arthritis development, affecting both neovascularization and the progression of experimentally induced synovitis.     

Identification of blood biomarkers of rheumatoid arthritis by transcript profiling of peripheral blood mononuclear cells from the rat collagen-induced arthritis model

Rheumatoid arthritis (RA) is a chronic debilitating autoimmune disease that results in joint destruction and subsequent loss of function. To better understand its pathogenesis and to facilitate the search for novel RA therapeutics, we profiled the rat model of collagen-induced arthritis (CIA) to discover and characterize blood biomarkers for RA. Peripheral blood mononuclear cells (PBMCs) were purified using a Ficoll gradient at various time points after type II collagen immunization for RNA preparation. Total RNA was processed for a microarray analysis using Affymetrix GeneChip technology. Statistical comparison analyses identified differentially expressed genes that distinguished CIA from control rats. Clustering analyses indicated that gene expression patterns correlated with laboratory indices of disease progression. A set of 28 probe sets showed significant differences in expression between blood from arthritic rats and that from controls at the earliest time after induction, and the difference persisted for the entire time course. Gene Ontology comparison of the present study with previous published murine microarray studies showed conserved Biological Processes during disease induction between the local joint and PBMC responses. Genes known to be involved in autoimmune response and arthritis, such as those encoding Galectin-3, Versican, and Socs3, were identified and validated by quantitative TaqMan RT-PCR analysis using independent blood samples. Finally, immunoblot analysis confirmed that Galectin-3 was secreted over time in plasma as well as in supernatant of cultured tissue synoviocytes of the arthritic rats, which is consistent with disease progression. Our data indicate that gene expression in PBMCs from the CIA model can be utilized to identify candidate blood biomarkers for RA.     

Identification of blood biomarkers of rheumatoid arthritis by transcript profiling of peripheral blood mononuclear cells from the rat collagen-induced arthritis model

Rheumatoid arthritis (RA) is a chronic debilitating autoimmune disease that results in joint destruction and subsequent loss of function. To better understand its pathogenesis and to facilitate the search for novel RA therapeutics, we profiled the rat model of collagen-induced arthritis (CIA) to discover and characterize blood biomarkers for RA. Peripheral blood mononuclear cells (PBMCs) were purified using a Ficoll gradient at various time points after type II collagen immunization for RNA preparation. Total RNA was processed for a microarray analysis using Affymetrix GeneChip technology. Statistical comparison analyses identified differentially expressed genes that distinguished CIA from control rats. Clustering analyses indicated that gene expression patterns correlated with laboratory indices of disease progression. A set of 28 probe sets showed significant differences in expression between blood from arthritic rats and that from controls at the earliest time after induction, and the difference persisted for the entire time course. Gene Ontology comparison of the present study with previous published murine microarray studies showed conserved Biological Processes during disease induction between the local joint and PBMC responses. Genes known to be involved in autoimmune response and arthritis, such as those encoding Galectin-3, Versican, and Socs3, were identified and validated by quantitative TaqMan RT-PCR analysis using independent blood samples. Finally, immunoblot analysis confirmed that Galectin-3 was secreted over time in plasma as well as in supernatant of cultured tissue synoviocytes of the arthritic rats, which is consistent with disease progression. Our data indicate that gene expression in PBMCs from the CIA model can be utilized to identify candidate blood biomarkers for RA.     

High synovial expression of the inhibitory FcgammaRIIb in rheumatoid arthritis

Activating Fc gamma receptors (FcgammaRs) have been identified as having important roles in the inflammatory joint reaction in rheumatoid arthritis (RA) and murine models of arthritis. However, the role of the inhibitory FcgammaRIIb in the regulation of the synovial inflammation in RA is less known. Here we have investigated synovial tissue from RA patients using a novel monoclonal antibody (GB3) specific for the FcgammaRIIb isoform. FcgammaRIIb was abundantly expressed in synovia of RA patients, in sharp contrast to the absence or weak staining of FcgammaRIIb in synovial biopsies from healthy volunteers. In addition, the expression of FcgammaRI, FcgammaRII and FcgammaRIII was analyzed in synovia obtained from early and late stages of RA. Compared with healthy synovia, which expressed FcgammaRII, FcgammaRIII but not FcgammaRI, all activating FcgammaRs were expressed and significantly up-regulated in RA, regardless of disease duration. Macrophages were one of the major cell types in the RA synovium expressing FcgammaRIIb and the activating FcgammaRs. Anti-inflammatory treatment with glucocorticoids reduced FcgammaR expression in arthritic joints, particularly that of FcgammaRI. This study demonstrates for the first time that RA patients do not fail to up-regulate FcgammaRIIb upon synovial inflammation, but suggests that the balance between expression of the inhibitory FcgammaRIIb and activating FcgammaRs may be in favour of the latter throughout the disease course. Anti-inflammatory drugs that target activating FcgammaRs may represent valuable therapeutics in this disease.     

Gene expression profiling and functional analysis of angiogenic markers in murine collagen-induced arthritis

Introduction

Dysregulated angiogenesis is implicated in the pathogenesis of rheumatoid arthritis (RA). To provide a more profound understanding of arthritis-associated angiogenesis, we evaluated the expression of angiogenesis-modulating genes at onset, peak and declining phases of collagen-induced arthritis (CIA), a well-established mouse model for RA.

Methods

CIA was induced in DBA/1 mice with type II collagen. Functional capillary density in synovial tissue of knee joints was determined by intravital fluorescence microscopy. To assess the ability of arthritic joint homogenates to induce angiogenesis, an endothelial chemotaxis assay and an in vivo matrigel plug assay were employed. The temporal expression profile of angiogenesis-related genes in arthritic paws was analysed by quantitative real-time RT-PCR using an angiogenesis focused array as well as gene specific PCR. Finally, we investigated the therapeutic effect of a monoclonal antibody specifically blocking the binding of VEGF to neuropilin (NRP)-1.

Results

Although arthritic paw homogenates displayed angiogenic activity in vitro and in vivo, and synovia of arthritic paws appeared highly vascularised on histological examination, the functional capillary density in arthritic knee synovia was significantly decreased, whereas capillary diameter was increased. Of the 84 genes analysed, 41 displayed a differential expression in arthritic paws as compared to control paws. Most significant alterations were seen at the peak of clinical arthritis. Increased mRNA expression could be observed for VEGF receptors (Flt-1, Flk-1, Nrp-1, Nrp-2), as well as for midkine, hepatocyte growth factor, insulin-like growth factor-1 and angiopoietin-1. Signalling through NRP-1 accounted in part for the chemotactic activity for endothelial cells observed in arthritic paw homogenates. Importantly, therapeutic administration of anti-NRP1^B antibody significantly reduced disease severity and progression in CIA mice.

Conclusions

Our findings confirm that the arthritic synovium in murine CIA is a site of active angiogenesis, but an altered balance in the expression of angiogenic factors seems to favour the formation of non-functional and dilated capillaries. Furthermore, our results validate NRP-1 as a key player in the pathogenesis of CIA, and support the VEGF/VEGF receptor pathway as a potential therapeutic target in RA.